Nucleic acids DNA and RNA consist of a sugar/phosphate backbone with attached linear sequence of bases: thymine (T), cytosine (C , guaninc (G) and adenine (A) in the case of DNA and C,G,A and uracil (U) in the case of RNA. The bases bind to one another as follows: EQU C-G EQU A-T/U
The sequence of bases determines the sequence of amino acids of protein produced from the nucleic acids, and mutations in nucleic acids result in variations in the sequences of bases and thereby amino acid sequence.
The definition of exact single base changes in genes as a result of mutation is an important goal in the study of genetics. As the sequencing of complete genes in search of base changes is tedious several attempts have been made to make the search more efficient (see references 1, 2, 3, 4,5). Heteroduplexes were formed between wild type and variant DNA and it was found that single strand specific SI nuclease could cleave the DNA at the point of the mismatched bases in the DNA (1). The differential mobility of native and denatured DNA/DNA heteroduplexes coupled with their differential melting temperatures has been exploited by Myers et al (2). Since this method was not generally applicable (reviewed in 3), Myers et al (3) described a method where mismatches in heteroduplexes between RNA and DNA were cleaved by ribonuclease A. An alternative approach where RNase A is used to cleave mismatches in RNA/RNA heteroduplexes has also been described (4). Finally Novak et al (5) have reported that single base pair mismatches in DNA/DNA heteroduplexes are reactive with, but are not cleaved by, a carbodiimide.
The known methods either do not detect and localise all mutations, or have not been shown to do so, and the present invention aims to provide an improved method, which in preferred embodiments at least, is capable of detecting all mutations.